Genetic risk of depression is different in subgroups of dietary ratio of tryptophan to large neutral amino acids

Manipulation of intake of serotonin precursor tryptophan has been exploited to rapidly induce and alleviate depression symptoms. While studies show that this latter effect is dependent on genetic vulnerability to depression, the effect of habitual tryptophan intake in the context of predisposing genetic factors has not been explored. Our aim was to investigate the effect of habitual tryptophan intake on mood symptoms and to determine the effect of risk variants on depression in those with high and low tryptophan intake in the whole genome and specifically in serotonin and kynurenine pathways. 63,277 individuals in the UK Biobank with data on depressive symptoms and tryptophan intake were included. We compared two subpopulations defined by their habitual diet of a low versus a high ratio of tryptophan to other large amino acids (TLR). A modest protective effect of high dietary TLR against depression was found. NPBWR1 among serotonin genes and POLI in kynurenine pathway genes were significantly associated with depression in the low but not in the high TLR group. Pathway-level analyses identified significant associations for both serotonin and kynurenine pathways only in the low TLR group. In addition, significant association was found in the low TLR group between depressive symptoms and biological process related to adult neurogenesis. Our findings demonstrate a markedly distinct genetic risk profile for depression in groups with low and high dietary TLR, with association with serotonin and kynurenine pathway variants only in case of habitual food intake leading to low TLR. Our results confirm the relevance of the serotonin hypothesis in understanding the neurobiological background of depression and highlight the importance of understanding its differential role in the context of environmental variables such as complexity of diet in influencing mental health, pointing towards emerging possibilities of personalised prevention and intervention in mood disorders in those who are genetically vulnerable.

 Supplementary Table S15. SNP level association results with respect to depressive symptoms concerning the serotonin pathway for low and high TLR subgroups, and for the total population. P-values are displayed for low and high TLR subgroups and also for the total population. This is provided as a part of the Supplementary text file F3 (compressed).
 Supplementary Table S16. SNP level association results with respect to depressive symptoms concerning the kynurenine pathway for low and high TLR subgroups, and for the total population. P-values are displayed for low and high TLR subgroups and also for the total population. This is provided as a part of the Supplementary text file F3 (compressed).
 Supplementary Table S17. SNP level association results with respect to depressive symptoms concerning all SNPs for the total population, and for low and high TLR subgroups. P-values are displayed for low and high TLR subgroups and also for the total population. This is provided as a Supplementary text file F3 (compressed).
 Supplementary Table S18. LNAA and tryptophan intake in low and high TLR groups.
Unit denotes the measurement unit for each variable as available in the UKB data.
Difference of means (absolute) and Difference of means (relative vs low TLR) denote the absolute and relative difference between means of low and high TLR subgroups. Relative differences are shown with respect to the means of low TLR subgroup. Difference of means (p-value) displays the p-value related to the t-test for difference of means between low and high TLR groups for each variable. Sig. marks significant p-values taking into consideration a correction for multiple testing with a threshold of 0.0025. Markers (*), (**), and (***) denote statistically significant difference, highly significant difference (p< 1.0e-6), and clear difference (p< 2.2e-16) respectively.
 Supplementary Table S19. Plasma CRP level in low and high TLR groups. Difference of means (absolute) and Difference of means (relative vs low TLR) denote the absolute and relative difference between means of low and high TLR subgroups. Relative difference is shown with respect to the mean of low TLR subgroup. Difference of means (p-value) displays the p-value related to the t-test for difference of means between low and high TLR groups.

Derivation of TLR
Assessing the protein content of various food types, more specifically their amino acid content, was the first step of computing TLR, i.e. tryptophan -large neutral amino acid (LNAA) ratio of a

QC methodology
The following QC steps were performed on the whole dataset. First, we removed samples the consent of which were withdrawn, were not of white British ancestry, or putatively had a sex chromosome aneuploidy (see Supplementary Figure S5). Second, only biallelic SNP variants were retained, from both the set of genotyped and imputed variants, furthermore, imputed variants with 'info' or 'certainty' values lower than 0.9 were also excluded.
Genotyped and imputed genotypic values were merged, with genotyped ones having precedence.
To exclude samples and variants which might be considered unreliable for the purpose of statistical tests, the following filtering steps were performed: (1) variants with minor allele frequency values lower than 0.01 were excluded (2) variants and samples with missing rates greater than 0.01 were excluded (this was done in an iterative manner, with more and more rigorous threshold values 0.1, 0.05 and 0.01, the filters alternatingly applied first to variants then to samples for each threshold value) (3) variants which could be assumed not to be in Hardy-Weinberg equilibrium with a p-value more relevant than (i.e. less than) 1e-5 were excluded The set of variants that was achieved after the above steps was regarded as the input of our association tests.
Samples were further filtered using the following steps: (4) Samples whose F-statistics (as calculated by the command '--check-sex' by plink) were not unambiguous were removed.
(5) Samples which were deemed outliers regarding their observed and expected autosomal homozygous genotype counts were removed.
(6) Finally, variants with a minor allele frequency less than 0.01 in either of the subpopulations were excluded.
Note: The QC used in BOLT-LMM and set-test does not include steps for kinship filtering because of these methods can handle related samples, hence there is less information loss in the process.

Relevant serotonin and kynurenine SNPs
The serotonin SNP rs10089380 located in the 10 kilobase neighbourhood of the RB1CC1 gene

Relevant SNPs not related to candidate pathways
Regarding SNPs outside of our candidate pathways, in the low TLR subgroup exclusively, 119 SNPs showed association with depressive symptoms with suggestive significance (p<1E-05).
Some of these SNPs are intergenic (76)

Relevant serotonin and kynurenine genes: NPBWR1 and POLI
NPBWR1 shows the highest expression levels in blood, liver and brain, especially in the cerebral cortex, hypothalamus and pituitary gland, according to the Human Protein Atlas v20.0 5 .
Neuropeptide W and neuropeptide B, ligands of the G protein-coupled receptor encoded by this gene, play a role in neuroendocrine regulation, energy homeostasis, feeding behaviour, circadian rhythm and sleep, emotions, as well as in pain sensation and cardiovascular functions (Chottova Dvorakova, 2018). In animal studies, NPBWR1 has been associated with stress response in new environments 6 , with social behaviour and fear processing 7 , and with metabolic disturbances and adult-onset obesity 8 , which traits are also highly related to depression. In human studies, polymorphisms in the NPBWR1 gene altered face emotion processing 9 , and in GWAS studies were associated with obesity-related variables 10,11 , age at menarche 12 , and response to antidepressant 13 . Thus, based on previous studies and our present results, NPBWR1 may be a good target for novel treatment strategies in antidepressive therapies.
In the kynurenine pathway, the canonical candidate genes encoding the rate limiting metabolic enzymes of tryptophan catabolismthe indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) 14 did not emerge as top genes for depressive symptoms. The only significant association in the low TLR subgroup was with the POLI (DNA polymerase iota) gene, which only survived kynurenine pathway-level but not all gene-level correction. There were no kynurenine associations in the high TRL subgroup or in the total cohort. POLI is connected to the kynurenine pathway via potential binding with kynurenine metabolites (3-Hydroxyanthranilic Acid and Quinolinic Acid) and is mainly expressed in endocrine tissues 5 , including the testis and ovary, and the brain, especially in the cortex, the pons and medulla. Its main function is to promote DNA repair. Interestingly, a recent multivariate genome-wide-association meta-analysis study focusing on well-being-related traits, such as life satisfaction, positive affect, neuroticism, and depressive symptoms, demonstrated a significant association with regulatory regions of POLI 15 .

Relevant genes not related to candidate pathways
Amongst all genes, ATP5C1, TAF3 showed significant and the KIN gene suggestive significant association with depressive symptoms in the low TLR subgroup (see Supplementary Table S6).

Relevant pathways
Results related to the subsets of kynurenine pathway indicated that the non-specific binders subset from CHEMBL was associated with depressive symptoms in the low TLR subgroup and in the total population with p-value: 0.017 and 0.0034 respectively, which however was not significant (using correction for kynurenine subpathways, p-value<0.0033). In addition, the kynurenine WikiPathway subset (denoted as Kynurenine WP) showed significant association with depressive symptoms in the total population with p-value: 4.7E-4.
For the serotonin set, subpathways from all main sources (WikiPathway, GO and Reactome) showed associations with depressive symptoms in the low TLR subgroup and also in the total population. The Reactome serotonin subpathway showed a significant association both in the low TLR subgroup (p-value: 0.0015) and in the total population (p-value: 0.0016) that survived correction for serotonin subpathways (p-value<0.0021). Furthermore, the Serotonin WikiPathway only showed a significant association in the low TLR subgroup (p-value: 0.0019), whereas Reactome GPCR downstream and Serotonin GO showed a significant association only in the total population (p-value 0.0013). Importantly, none of the main subpathways was significant in the high TLR subgroup (for details see Supplementary Table S7).

Relevant GO terms
In the whole population, the most significant pathway was "adult behaviour" and several pathways related to brain development. In the low TLR subgroup, the most significant pathways associated with depression included "adaptive thermogenesis" (GO:1990845), "cerebral cortex cell Focusing on mechanisms significantly associated with depression in the low dietary TLR subgroup, "adaptive thermogenesis" pathway (GO:1990845) is related to regulated heat production in response to short-term environmental changes including for example stress, diet, or reduced temperature, and is implicated in reduced body weight maintenance and unsuccessful weight loss 16 . Thus adaptive thermogenesis may be clinically relevant with respect not only to diet but also depression, considering that depression is a stress-related condition associated with weight changes in either direction, which, especially weight gain, may play a crucial role in the negative consequences of depression on somatic health especially increased cardiovascular risk 17,18 and metabolic comorbidities in depression 19 . Several of the other most significant pathways in the low dietary TLR subgroup reflect aspects of neurodevelopment. The "cerebral cortex cell migration pathway" (GO:0021795), controlling key morphogenic events involved in neocortical neurodevelopment contributing to a precise spatial and temporal distribution of neural cells and also their maturation and formation of neural circuits has been implicated in neuropsychiatric disorders including first episode psychosis or schizophrenia 20 , susceptibility to juvenile myoclonic epilepsy 21 , or autism spectrum disorder associated with 16p11.2 copy number variation 22 .
"Negative regulation of ERBB signalling pathway" (GO:1901185) via neuregulin/ERBB signalling also plays a role in neurodevelopment via regulating the assembly of neural circuitry playing a role also in synaptic plasticity, myelination and neurotransmission, with abnormal signalling contributing to impaired brain function and to susceptibility of neuropsychiatric disorders including depression, bipolar disorder or schizophrenia 23 . Besides multiple and in part overlapping pathways and mechanisms significantly associated with depression in the low dietary TLR subgroup and the total population, in the high dietary TLR subgroup there was a remarkable relative lack of significantly associated pathways, with only general and not solely brain-relevant mechanisms such as "regulation of calcium ion membrane transport" (GO:1903169) and "negative regulation of peptidyl serine phosphorylation" (GO:0033137) showing an association, and none of the mechanisms that showed association in the low dietary TLR subgroup or the whole sample were significant in the high TLR subgroup.
Besides "cerebral cortex cell migration pathway" and "negative regulation of ERBB signalling pathway", the GO term "regulation of neural precursor cell proliferation" (GO:2000177) is also associated with brain development. Neural precursor cells, including neural stem cells and neural progenitor cells, are temporally regulated cells with the ability for generating all neural cell types present in the brain via proliferation and differentiation into astrocytes, oligodendrocytes and mature neurons in the presence of growth factors. Especially adult neurogenesis has attracted significant attention as implicated in human neuropsychiatric disorders and also possibly in their treatment, although results are conflicting [24][25][26][27] . Considering that mechanisms related to brain development were significant only on the low TLR subgroup or the total sample but not the high TLR subgroup, our results suggest that the effect of such risk variants may be a function of diet, keeping in mind that several of the above processes reflect early neurodevelopment unaffected by current dietary habits. Interestingly, the most significant pathways in the whole population (in addition to being significant also in the low TRL subgroup) included "Blood vessel endothelial cell migration" (GO:0043534), related to angiogenesis which may be relevant in several ways. On the one hand, considering that blood vessels constitute key components of stem cell niches also in the adult brain controlling neuronal stem cell function 28 , this finding further supports the suggestion that TLR intake may be associated with depression via influencing neurogenesis.
Furthermore, blood vessel endothelial cell migration may also be related to cardiovascular risk and thus be important in the context of the increased cardiovascular comorbidity and mortality observable in depression 17,18 . The other most significant pathway in the total population was "response to organophosphorous" (GO:0046683) which may have relevance with respect to dementia, considering the role of organophosphate acetylcholinesterase inhibitors employed in the treatment of Alzheimer's disease and other dementias, and also the association with depression with earlier onset of cognitive decline 29 .

Addition to the rational of the study: effects of acute tryptophan depletion challenge in human and animal studies
Previous studies demonstrated that ATD induces emotional, cognitive and memory abnormalities only in individuals with susceptibility to depressive and anxiety disorders [30][31][32] and had no or minimal effect on mood in healthy individuals without family history for psychiatric disorders 33 .
Furthermore, animal studies showed that after ATD the decrease in 5-HT synthesis is not ubiquitous in the brain, it is more effective in the hippocampus, but also relevant in frontal and prefrontal cortex. In addition, these effects were more prominent in mice strain with impaired 5-HT synthesis and a more anxious phenotype 34 . In humans, ATD has not changed the default mode network (DMN) connectivity in healthy volunteers, despite that plasma TRP level decreased significantly, which is especially interesting as DMN connectivity was associated with depressive symptoms 35 . Therefore, in this study, our aim was to provide explanation for differential susceptibility to the development of depressive symptoms in those with habitual low dietary ratio of TRP/LNAA and to identify which genetic risk factors contribute to depression in case of low or high dietary ratio of TRP/LNAA. Based on previous negative depression GWAS studies regarding the classical canonical serotonin and kynurenine related genes we would expect perturbation of the downstream signalling mechanisms and regulatory biological processes of these candidate pathways.